Probes for identifying cancer-specific antigens

ABSTRACT

This invention relates to the diagnosis of cancer, and in particular to the identification and detection of cancer-specific antigens, the invention provides antibody probes and methods for using the probes for detection and purification of cancer-specific antigens, and in the preparation of vaccine compositions. Antibodies against cancer-specific antigens are also disclosed and claimed.

This invention relates to the diagnosis of cancer, and in particular tothe identification and detection of cancer-specific antigens. Theinvention provides antibody probes and methods for using the probes fordetection and purification of cancer-specific antigens, and in thepreparation of therapeutic, diagnostic and vaccine compositions, as wellas in the preparation of antibodies directed against cancer-specificantigens.

BACKGROUND OF THE INVENTION

All references, including any patents or patent applications, cited inthis specification are hereby incorporated by reference. No admission ismade that any reference constitutes prior art. The discussion of thereferences states what their authors assert, and the applicants reservethe right to challenge the accuracy and pertinency of the citeddocuments. It will be clearly understood that, although a number ofprior art publications are referred to herein, this reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art, in Australia or in any othercountry.

Considerable effort has been devoted to the search for therapies anddiagnostics for cancers. The identification of cancer-specific moleculeshas remained the major stumbling block to the generation of suitable andeffective therapies and diagnostics. One of the major reasons for thisis the similarity between cancer cells and normal cells. It is possiblethat there are only very subtle variations in the proteins encodedduring the formation of cancer cells. The search for these subtlevariations is often driven by hope or inspiration, and entailsconsiderable difficulty, expense and time.

Lung, colon and breast cancers are among the most common malignanttumours in humans. While methods of treatment have vastly improved inrecent years, the effectiveness of treatment is critically dependent onearly diagnosis. For example, while 70 to 80% of early detected, lymphnode-negative breast cancer patients survive over 10 years after primarytherapy, more than 60% die in this period if the tumour cells havealready reached the lymph nodes before the commencement of treatment.The most efficient methods of early diagnosis for breast cancer areself-examination and mammography. No such method of early diagnosis isavailable for lung cancers, which can only be detected by radiography,which is usually carried out after symptoms become apparent, resultingin a much lower ten-year survival rate (approximately 13%). It istherefore generally accepted that improved, simple methods of earlydiagnosis would have a profound effect on the outcome of cancertreatments and mortality.

The most commonly used technique in clinical diagnosis in bothveterinary and human medicine is the detection of antibodies or antigensin the serum of patients. The usefulness of serological tests for cancerdiagnosis has however been disappointing, partly because cancer-specificor cancer-associated antigens have not been well defined andcharacterised. In addition, the use of serum as a source of specificantibodies has several inherent disadvantages, including:

-   -   1. The presence of large amounts of serum antibodies not related        to the pathological agent, resulting in false positive results        and background reactions;    -   2. The formation of antigen-antibody complexes in the serum        which can disguise the presence of low titre        antibodies/antigens;    -   3. The inability to detect antibodies produced locally at        restricted mucosal sites, such as breast, colon and lung        tissues, in the serum because of the massive dilution of these        antibodies upon movement into the blood; and    -   4. The likelihood of persistence of specific antibodies in the        serum, even in the absence of current disease makes it difficult        to differentiate between previous and current disease.

Prior art methods have primarily been directed to generation ofmonoclonal antibodies directed to cancer-specific antigens. For example,U.S. Pat. No. 5,093,261 by Hagiwara et al. discloses the generation ofhuman hybridoma cell lines which produce monoclonal antibody specificfor a primary liver cancer by fusion of lymph node lymphocytes from apatient with liver cancer with cells of a lymphoblastoid cell line.However, such methods are directed to making monoclonal antibodiesagainst an unidentified antigen of a known cancer, rather than toidentifying a cancer-specific antigen. In addition, monoclonal antibodyproduction requires complex in vitro fusion and selection proceedings;the monoclonal antibodies thus produced do not reflect the totalpolyclonal immune response mounted against the cancer by the B cells inthe draining lymph nodes. There is no disclosure or suggestion that themethods disclosed therein could be applied to preclinical diagnosis,staging of cancers, or detection of metastasis.

Therefore it was an objective of the inventors to develop methods foridentifying antigenic molecules, or parts thereof, which are specificfor individual cancers. Such molecules would be useful in thedevelopment of diagnostic or therapeutic agents for specific cancers.

We have previously shown that it is possible to identify protectiveantigens which are specific for pathogens such as parasites or bacteriaof veterinary importance by culturing antibody-secreting cells fromdraining lymph nodes adjacent to a site of infection, isolatingimmunoglobulins from the culture medium, and using these immunoglobulinsto identify pathogen-specific antigens. See for example U.S. Pat. No.5,650,154; Meeusen and Brandon 1994a,b; and Walker et al, 1994, theentire disclosures of which are incorporated herein by this reference.

This method, which we have designated “ASC-probe technology”, utilisesantibody-secreting cells as the source of the antibodies, and has beenused extensively and successfully in the identification of newstage-specific and tissue-specific antigens and antibodies in endo- andecto-parasite, bacterial and mycoplasmal infections (Meeusen and Brandon1994a, b; Walker et al, 1994; Walker et al, 1996; Bowles et al, 1995;Bowles et al, 1996). This method also makes use of the versatility ofantibodies to identify and detect antigens by commonly used methods,such as Western blotting and immunoprecipitation. The source of theantibodies is not serum but antibody-secreting cells (ASC). It has longbeen established that ASCs are induced in the local lymph nodes draininga disease-affected tissue, where antigen is deposited. From the lymphnodes, activated lymphocytes, including ASCS, migrate via the efferentlymphatic vessel through the bloodstream to the target tissue. The ASCswithin the lymph nodes are short-lived, surviving for 4-6 days, and areonly present as long as the antigenic stimulus is present within thetissue.

In addition, we have shown that the specific ASCs are restricted to thelymph nodes draining the affected organ or tissue, and that differentlymph nodes within the same animal can react independently to differentstages of infection with a pathogen, generally with different isotypeand antigen recognition profiles (Meeusen and Brandon 1994b).

When cultured in vitro, the ASCs isolated from infected tissue ordraining lymph nodes can be induced to secrete high levels of specificantibodies into the culture supernatant for several days. Theantibody-containing supernatant, which we refer to as “ASC-probes”, isused directly to detect the presence and variety of antigens present ata particular time and tissue site and stage of infection. In addition topreparing ASC-probes from infected tissues and draining lymph nodes, wehave also been able to isolate specific ASC-probes from circulatingblood by making use of the narrow window of opportunity when ASCs aremigrating from the lymph nodes to the tissue via the blood circulation.We have found that ASC-probes have distinct advantages over the use ofserum antibodies for the discovery of novel pathogen-specific antigensuseful for diagnosis and vaccine development.

In addition, the use of ASC-probes overcomes the major disadvantages ofusing serum antibodies mentioned above, in that:

1. ASCs are only generated after antigen stimulation, and the antibodiespresent in the ASC-probes are therefore predominantly specific for thedisease agent, significantly reducing background and non-specificreactions;

2. As the cells used for ASC-probe preparation are washed free of serumbefore culture, there is no antigen present, and no antigen-antibodyreaction which can reduce the sensitivity of the assay can occur;

3. ASCs locally produced in mucosal tissues can be isolated from tissueor from lymph nodes, or from peripheral blood during their migration totissues;

4. As ASCs in lymph nodes are short-lived, they are only present as longas the antigenic stimulus is present in the tissue; the production ofASC-probes therefore reflects current infection or disease; and

5. ASC-probes, including those present in or secreted by peripheralblood lymphocytes, can provide a positive diagnosis, even at a stagewhen circulating antibody cannot be detected.

Since the development of this technique, there has been one publicationreporting the use of peripheral blood ASCs for the detection of HIVinfection in seronegative patients (Jehuda-Cohen et al. 1990).

While the use of ASC-probes has proven to be a major breakthrough inresearch relating to vaccines against infectious diseases(Meeusen andMaddox, 1999), this technique has not yet been applied to cancerresearch. The pathogenesis of cancers is very different from that ofparasitic or bacterial infections, and cancer-specific protectiveresponses are primarily T cell rather than B cell responses. Severalstudies have however reported the presence of activated B cells andplasma cells within draining lymph nodes and infiltrating lymphocytes ofbreast cancers (see for example Lynch and Houghton, 1993). However,there is no evidence as to whether this represents a specific responseto a cancer-associated antigen, and the nature of these antigens has notbeen identified. Despite this, we believed that it was possible thatcancer antigens might be specifically recognised by lymphocytes fromadjacent lymphoid tissues. We therefore wished to determine whether theASC-probe approach could also be successfully applied to cancerresearch, and whether ASC-probes could be used for the detection ofnovel tissue-specific and cancer-associated antigens, and for the earlydiagnosis of primary or recurrent cancer.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a method for producing anantibody-secreting cell probe (ASC-probe) against a cancer-specificantigen, comprising the steps of:

(a) obtaining a biological sample from an animal suffering from acancer;

(b) isolating a population of cells from the biological sample;

(c) culturing the cells in vitro in a suitable culture medium; and

(d) harvesting antibodies produced by lymphoid cells present in the cellpopulation.

The cancer may be a solid tumour, such as a cancer of the breast, ovary,uterus, prostate, colon, or lung, or a lymphoma, or may be a leukemia.

The cancer specific antigen may be a polypeptide or peptide, aglycoprotein, an oligosaccharide, a polysaccharide, or a glycolipid.However, it will be appreciated that this does not exclude thepossibility that the antigen may have some other chemical structure. Allthat is required is that the antigen is capable of eliciting antibodyproduction.

It will be clearly understood that the ASC probe may be of any antibodyisotype. In particular, while in many cases the ASC probe will be anIgG, the invention specifically encompasses ASC-probes which are IgA.

The animal from which the biological sample is taken may be a mammal,including humans. The term mammal includes companion animals such asdogs and cats, and domestic animals such as horses, cattle and sheep, orzoo animals such as felids, canids, bovids, and ungulates.

The biological sample may be of any suitable type, for example spleen,blood, lymph or lymph node, or bone marrow, but is preferably from atissue area which is rich in lymphoid cells and is close to the site ofthe cancer, such as a draining lymph node. In the case of leukemias, thebiological sample is preferably spleen or bone marrow.

In general blood samples are not preferred, because it has been foundthat the majority of antibodies found in a serum or plasma sample areirrelevant to the cancer. In addition, serum or plasma components mayinterfere with the specific reactions between cancer antigens and thecorresponding antibodies. In contrast, the ASC-probes of the inventionare highly enriched in cancer-specific antibodies. However, it will beappreciated that for some conditions such as leukemias it may benecessary to use blood as a source of antibody-secreting cells.

The cells isolated from the biological sample preferably include Blymphocytes and/or B memory cells. Optionally the sample is subjected toone or more processes for enrichment of lymphocytes or B lymphocytes.Suitable processes for such enrichment are well known in the art; seefor example Goding, 1986. Cell populations which may suppress antibodysecretion, in particular CD8⁺ T cells, may optionally be selectivelydepleted, for example by complement-mediated lysis of selected cellpopulations with monoclonal antibodies (mAbs) against CD5, CD8, CD4and/or γδ-TCR (Kamath et al., 2000).

Because the level of antibody secretion into the culture medium may bevery low without in vitro stimulation of resting lymphocytes, the methodpreferably includes a further step of activating the isolated cells toproliferate and to secrete antibodies, by adding a cell activating agentor a cell proliferation agent to the culture medium. In vitro secretionof antibodies into the culture medium by recently activated B cells mayalso be enhanced by the addition of mitogens or helper factors to thecultures.

The cell activating agent is preferably selected from the groupconsisting of mitogens and helper factors produced by leukocytes, theirsynthetic equivalents, or combinations thereof.

The mitogen is preferably selected from the group consisting of pokeweedmitogen (PWM), polyvinylpyrrolidone (PVP), phytohemagglutinin (PHA),Concanavalin A (Con A), CD40 ligands (see Banchereau et al., 1994)polyadenylic-polyuridylic acid (poly(A-U)), purified protein derivate(PPD), polyinosinic-polycytidylic acid (poly(I-C), lipopolysaccharide(LPS), staphylococcal organisms or products thereof, Bacto-streptolysinO reagent (SLO), staphylococcal phage lysate (SPL), Epstein-Barr virus(EBV), Nocardia water-soluble mitogen (NWEM), and dextran sulphate, oris a mixture of two or more of these agents.

The cell proliferation agent may be any agent which indirectly ordirectly results in B cell proliferation and/or antibody secretion, suchas solid-phase anti-immunoglobulin.

The helper factor may be a cytokine, such as an interleukin, a colonystimulating factor, an interferon, or any other helper factor which hasbeen shown to have a stimulatory effect on specific B cell proliferationand/or antibody secretion, either alone, or in combination with otherfactors and agents. Interleukin 6 is preferred.

It will be appreciated that cell activating agents, cell proliferationagents, mitogens or helper factors may be used separately or incombination, and that one or more of each of the additives may be used.

The ASC-probes are suitably harvested by separating the supernatant fromthe cells by centrifugation. The supernatant contains antibodiessecreted by the cells during culture, or released from the B cells, forexample by lysis of the B cells.

In a second aspect, the invention provides an ASC-probe directed againsta cancer-specific antigen; preferably the ASC is prepared using themethods of the invention. In one preferred embodiment of this aspect ofthe invention, the ASC probe is coupled to a solid support, which ispreferably one suitable for affinity chromatography, as discussed below.In a second preferred embodiment, the ASC probe is labelled with adetectable marker, such as a radioactive, fluorescent, enzymic, orchemiluminescent label; suitable such labels are well known to thoseskilled in the art, and the coupling may be performed by conventionalmethods. Biotin is a particularly preferred label.

The ASC-probes may be utilized simply in the form of the supernatantharvested from the cultured cells. Alternatively, the antibodies may beseparated and purified. The ASC-probes can be purified by conventionalmethods used to purify immunoglobulins from serum or plasma or fromtissue culture medium, such as precipitation with ammonium sulphate,fractionation with caprylic acid, ion exchange chromatography, orbinding to and elution from immobilized anti-immunoglobulin, protein Gor protein A.

Accordingly, in a third aspect the invention provides a method ofisolating an antigen associated with a cancer, comprising the steps of:

(a) obtaining a tissue or cell sample from a cancer;

(b) reacting the sample with an ASC-probe according to the invention,thereby to detect at least one antigen; and

(c) isolating the antigen thus detected.

It will be appreciated that the cancer-associated antigen may bespecific to a particular cancer, or alternatively may not be specific,but may nevertheless be useful in diagnosis and/or treatment of thecancer.

The cell or tissue sample may suitably be mixed with a standard buffersolution and placed on a standard support such as an SDS-polyacrylamidegel to separate the proteins present therein by electrophoresis. Theseparated proteins may then be transferred to nitro-cellulose, nylon orother sheets.

The step of reaction with an ASC-probe preferably further includesdetecting the product thus produced, using a detection assay such asWestern blotting, immunoprecipitation assay, a radioimmunoassay, anenzyme-linked immunoassay, chemiluminescent assay or immunofluorescentassay.

The ASC-probe of the invention may be used for the affinity purificationof a cancer-specific antigen. Thus in a third aspect the inventionprovides a method for purifying a cancer-specific antigen, comprisingthe steps of:

(a) subjecting a crude antigen mixture present in an extract of cancercells or cancer tissue to affinity chromatography using an ASC-probeaccording to the invention, immobilized on a suitable support, and

(b) isolating antigen bound to the immobilised ASC-probe.

The ASC-probe of the invention can be immobilised by coupling it to anysuitable solid support, e.g. CNBr-activated Sepharose 4B (Pharmacia),Affi-gel (Bio-RAD) or other affinity chromatography supports able tobind proteins, using the methods recommended by the manufacturer. Theimmobilized ASC-probe can then be used for the fractionation andpurification of specific antigen from a complex dell or tissue extractby affinity chromatography. After binding of antigen to immobilizedASC-probe, unbound or loosely bound macromolecular species can be washedaway from the solid support with a buffer, for example a buffercontaining 1.5M NaCl. Subsequently the antigen can be eluted from theaffinity column with low or high pH buffer or a buffer containingchaotropic ions, such as 0.5-3.0M sodium thiocyanate.

The application of the ASC-probe of the invention to affinitychromatography enables sufficient quantities of cancer-specific antigensto be rapidly isolated from a complex crude extraction mixture forbiochemical characterization, amino-acid sequencing and investigation ofthe ability of the antigens to elicit a protective immune response. Theuse of affinity chromatography for obtaining antigen(s) avoids thedifficulties often encountered when applying conventional biochemicaltechniques to the purification of an antigen about which little or nodata is known. It also obviates the need to raise polyclonal ormonoclonal antibodies for the purpose of analytical affinitychromatography. Large-scale preparation may, however, require thepreparation of polyclonal or monoclonal antibodies.

Having thus identified a cancer-specific antigen, where the antigen is apolypeptide or peptide, conventional molecular biology techniques suchas cloning, or chemical techniques such as solid phase polypeptidesynthesis may be used to produce large amounts of the antigen. Peptidescorresponding to different epitopes of the antigens may be used toproduce a vaccine. It will be clearly understood that thecancer-specific antigen may also be a glycoprotein or anoligosaccharide, a polysaccharide, a glycolipid, or some other chemicalstructure.

Accordingly in a fifth aspect the invention provides a method forpreparing a cancer-specific polypeptide or peptide antigen, comprisingthe steps of:

(a) providing a DNA library derived from a sample of a cancer;

(b) generating polypeptides from the library;

(c) probing the polypeptides with an antibody probe selected from thegroup consisting of an ASC-probe according to the invention, amonoclonal antibody derived therefrom, or a derivative of such amonoclonal antibody;

(d) identifying cDNA or genomic clones which produce polypeptides whichreact with the antibody probe; or

(e) probing the cDNA or genomic library with synthetic oligonucleotideprobes based on the amino acid sequence of the antigen identified andpurified as described above; and

(f) isolating the clones thus detected.

Either a cDNA library or a genomic library may be used. The library maybe assembled into suitable expression vectors that will enabletranscription and the subsequent expression of the cloned DNA, either inprokaryotic hosts such as bacteria or eukaryotic hosts such as yeast ormammalian cells.

The antibody probes in step (c) are preferably selected from:

(i) antibodies obtained from the culture medium produced as describedabove;

(ii) monoclonal or polyclonal antibodies produced against an antigenidentified and purified as described above;

(iii) recombinant or synthetic monoclonal antibodies or polypeptideswith specificity for an antigen identified and purified as describedabove, e.g. antibodies prepared as described by Ward et al (1989); and

(iv) single chain antibodies produced against an antigen identified andpurified as described above.

It will be clearly understood that the antibodies of (iii)-(v) may beproduced using a recombinant antigen according to the invention, or anantibody which has been derived from cDNA extracted from lymphocytes.Methods for the latter are described in U.S. Pat. No. 5,627,052 bySchrader.

In one preferred embodiment the antigen is able to elicit a protectiveantibody against a cancer. Protective antibodies can be identified bytheir ability to cause regression of the cancer and/or to delayprogression of the cancer, using methods known in the art.

In another preferred embodiment the antigen is associated with aspecific stage of development of the cancer. Such stage-specificantigens are useful in diagnostic staging of cancers in individualpatients.

In a third preferred embodiment the antigen is able to elicit lymphocyteproliferation and/or production of cytokine by antigen-specificlymphocytes; this may readily be assessed using methods known in theart.

In a sixth aspect the invention provides a polyclonal or monoclonalantibody directed against a cancer-specific antigen according to theinvention. Such antibodies may be used in diagnostic tests, or forpassive treatment of the cancer. It will be appreciated that polyclonalor monoclonal antibodies and fragments or analogues of monoclonalantibodies, such as ScFv fragments, humanized monoclonal antibodies,bispecific or chimeric antibodies are within the scope of the invention;methods for production of such polyclonal or monoclonal antibodies,fragments or analogues are well known in the art.

In a seventh aspect the invention provides a therapeutic or diagnosticcomposition, comprising a cancer-specific antigen according to theinvention, together with a physiologically or diagnostically acceptablecarrier. Where the composition is a diagnostic composition the antigenmay be specific to a particular stage of the cancer.

In an eighth aspect the invention provides a composition comprising atherapeutically effective amount of at least one antibody or specificligand which binds to a cancer-specific antigen identified according tothe invention, together with a pharmaceutically acceptable carrier.Preferably the antibody is a monoclonal or recombinant antibody whichbinds specifically to the antigen. The specific ligand may be a lectinor a peptide selected for specific binding to the antigen, for exampleby panning of a peptide library. This and other suitable methods ofselection are well known in the art.

In a ninth aspect the invention provides a method for the treatment ofcancer in an animal, comprising the step of administering atherapeutically effective amount of an antibody or specific ligand to acancer-specific antigen produced according to methods of the inventionto an animal in need of such treatment. Preferably the antibody is amonoclonal antibody. The antibody or ligand may optionally be linked toa toxin such as diphtheria toxin or ricin or to a chemotherapeuticagent, and used to target the toxin or agent to the tumour cells.

In a tenth aspect the invention provides a vaccine composition against acancer, comprising a prophylactically or therapeutically effectiveamount of at least one protective antigen prepared by a method of theinvention, or an antigenically-active antigen fragment thereof,optionally together with an adjuvant.

The vaccine or composition according to the invention may beadministered orally, or may be administered parenterally, for example byintramuscular, subcutaneous or intravenous injection. The amountrequired will vary with the antigenicity of the active ingredient, andneed only be an amount sufficient to induce an immune response typicalof existing vaccines, which may readily be determined by routineexperimentation. Typical initial doses will be approximately 0.001-1 mgactive ingredient/kg body weight. The dose rate may be increased, ormultiple doses may be used as needed to provide the desired level ofprotection.

The vaccine or composition according to the present invention mayfurther include a pharmaceutically acceptable carrier, diluent orexcipient therefor. Preferably the active ingredient is suspended ordissolved in a physiologically acceptable carrier. The carrier may beany solid or solvent which is non-toxic to the animal and compatiblewith the active ingredient. Suitable carriers include liquid carriers,such as normal saline and other non-toxic salts at or near physiologicalconcentrations, and solid carriers, such as talc or sucrose. Adjuvants,such as complete or incomplete Freund's adjuvant, alum, or iscoms, orimmunomodulators such as cytokines may optionally be added to enhancethe antigenicity of the antigen. When used for administration to thelungs, the vaccine composition is suitably in the form of an aerosol.

Methods and pharmaceutical carriers for preparation of pharmaceuticalcompositions are well known in the art, as set out in textbooks such asRemington's Pharmaceutical Sciences, 19th Edition, Mack PublishingCompany, Easton, Pa., USA.

For the purposes of this specification it will be clearly understoodthat the word “comprising” means “including but not limited to”, andthat the word “comprises” has a corresponding meaning.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram illustrating the use of ASC probes fordetection of cancer-associated antigens.

FIG. 2 shows the comparative levels of protein G-purified, biotinylatedantibody isolated from ASC culture supernatant from breast lymph nodecell cultures. This antibody was used for probing the Western blots inFIGS. 3 and 4. Purified antibody from culture supernatant is shown inlane 1, and antibody purified from control serum is shown in lane 2.

10 μl of biotinylated antibody at 1/150 and 1/500 dilution respectivelywas loaded on a 10% SDS-PAGE gel, transferred to nitrocellulose,incubated with peroxidase-conjugated streptavidin and developed byenhanced chemiluminescence.

FIG. 3 shows the results obtained with samples from breast cancerpatients, using Western blotting on nitrocellulose from a 10% SDS-PAGEgel.

Samples were solubilized in non-reducing SDS sample buffer.

Lane 1: Breast tumour tissue probed with biotinylated antibody purifiedfrom ASC-cultures.

Lane 2: Breast tumour tissue probed with biotinylated antibody purifiedfrom control serum.

Lane 3: Normal breast tissue probed with biotinylated antibody purifiedfrom ASC-cultures.

Lane 4: Normal breast tissue probed with biotinylated antibody purifiedfrom control serum.

All tissues and the ASC-probe were obtained from the same patient.

FIG. 4 shows results obtained using samples of urine from breast cancerpatients. Samples were subjected to Western blotting on nitrocellulosefrom a 10% SDS-PAGE gel. Samples were solubilized in non-reducing SDSsample buffer. All strips were probed with ASC culture supernatant.

Lane 1: Urine from breast cancer-positive patient 1

Lane 2: Urine from breast cancer-positive patient 2

Lane 3: Urine from breast cancer-negative patient 1

Lane 4: Urine from breast cancer-negative patient 2.

FIG. 5 is a schematic representation illustrating the evaluation ofperipheral blood ASC-probes for cancer diagnosis.

FIG. 6 is a schematic representation illustrating the diagnosticevaluation of peripheral blood and urine for cancer-specific antibodies.

FIG. 7 is a schematic representation illustrating the diagnosticevaluation of peripheral blood and urine for ASC-probe positiveantigens.

FIG. 8 shows the result of Western blot analysis of normal breast tissueand breast tumour tissue probed with biotinylated antibody obtained frombreast-draining lymph nodes. No: Normal tissue; Tu: tumour tissue.Molecular weight standards: 250,000; 98,000; 64,000; 50,000; 36,000;30,000. The arrows indicate regions of difference between samples fromnormal and tumour tissue.

FIG. 9 shows the results of Western blot analysis of normal ovariantissue and ovarian tumour tissue. No: Normal tissue; Tu: tumour tissue.Molecular weight standards: 250,000; 98,000; 64,000; 50,000; 36,000;30,000. The arrows and brackets indicate regions of difference betweennormal tissue and tumour tissue.

FIG. 10 shows the results of Western blot analysis of normal prostatetissue and prostate tumour tissue probed with biotinylated antibodyobtained from prostate-draining lymph nodes of patient A. No: Normaltissue; Tu: tumour tissue. Molecular weight standards: 250,000; 98,000;64,000; 50,000; 36,000; 30,000. The brackets indicate the regions ofdifference between the normal tissue and the tumour tissue.

FIG. 11 shows the results of protein staining and Western blot analysisof immunoprecipitated normal ovarian tissue and ovarian tumour tissueprobed with biotinylated antibody obtained from ASC-probes generatedfrom the draining lymph nodes of the same patient. PS: Coomassie Bluestaining. No: Normal tissue; Tu: tumour tissue. Molecular weightstandards: 250,000; 98,000; 64,000; 50,000; 36,000; 30,000. The arrowsand brackets indicate regions of difference between normal tissue andtumour tissue.

FIG. 12 is a schematic illustration showing how background caused byendogenous IgG in the tissue may be blocked by pre-incubation withmonomeric anti-IgG F(ab).

FIG. 13 is a schematic illustration showing how background caused byendogenous IgG can be suppressed by pre-incubation of ASC antibody withexcess biotinylated F(ab) fragments of anti-IgG, followed by excessnormal IgG.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail by way of reference onlyto the following non-limiting examples and to the drawings.

Monoclonal antibodies directed toward the cancer-specific antigen of theinvention are produced using any method which provides for theproduction of antibody molecules by continuous cell lines in culture.The word “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. Examples of suitable methods for preparing monoclonalantibodies include the original hybridoma method of Kohler, et al.,1975, and the human B-cell hybridoma method, Kozbor 1984; Brodeur etal., 1987.

The monoclonal antibodies of the invention specifically include“chimeric” antibodies (immunoglobulins) in which a portion of the heavyand/or light chain is identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (Cabilly, et al., U.S. Pat.No. 4,816,567; Morrison, et al., 1984).

In a preferred embodiment, the chimeric antibody is a “humanized”antibody. Methods for humanizing non-human antibodies are well known inthe art. Generally, a humanized antibody has one or more amino acidresidues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.

Humanization can be performed following methods known in the art (Jones,et al., 1986; Riechmann, et al., 1988; Verhoeyen, et al. 1988), bysubstituting rodent complementarity-determining regions (CDRs) for thecorresponding regions of a human antibody. Alternatively, it is nowpossible to produce transgenic animals (e.g., mice) that are capable,upon immunization, of producing a full repertoire of human antibodies inthe absence of endogenous immunoglobulin production. For example, it hasbeen described that the homozygous deletion of the antibody heavy-chainjoining region (JH) gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. See, for example, Jakobovits, et al., 1993; Jakobovits, etal., 1993; Bruggermann, et al. 1993. Human antibodies can also beproduced in phage-display libraries (Hoogenboom, et al., 1991; Marks, etal., 1991).

For diagnostic applications, the antibodies typically will be labeledwith a detectable moiety. The detectable moiety can be any one which iscapable of producing, either directly or indirectly, a detectablesignal. For example, the detectable moiety may be a radioisotope, suchas ³H, ¹⁴C ³²P, ³⁵S, or ¹²⁵I, a fluorescent or chemiluminescentcompound, such as fluorescein isothiocyanate, rhodamine, or luciferin;radioactive isotopic labels, such as, e.g., ¹²⁵I, ³²P, ¹⁴C, or ³H, or anenzyme, such as, alkaline phosphatase, betagalactosidase or horseradishperoxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed, including those methods describedby David et al., 1974; Pain et al., 1981; and Bayer et al. 1990.

The antibodies may be employed in any known assay method, such ascompetitive binding assays, direct and indirect sandwich assays, andimmunoprecipitation assays (Zola, 1987).

Competitive binding assays rely on the ability of a labeled standard(e.g., the cancer-specific antigen or an immunologically reactiveportion thereof) to compete with the test sample analyte (thecancer-specific antigen) for binding with a limited amount of antibody.The amount of cancer-specific antigen in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See for example David, et al., U.S. Pat. No.4,376,110. The second antibody may itself be labeled with a detectablemoiety (direct sandwich assays) or may be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assay). For example, one type of sandwich assay is anELISA assay, in which case the detectable moiety is an enzyme.

Methods for preparation of divalent and trivalent scFc-type constructsare respectively described in PCT/AU93/00491 and PCT/AU98/00212 byCommonwealth Scientific and Industrial Research Organisation.

Collection of Human Tissue and Cells

Lymphocytes were isolated primarily from lymph nodes, biopsies,peripheral blood and resected cancerous tissue removed during surgery ofpatients. Samples of tissue from tumours at different stages of growthwere obtained from patients after surgery, and classified according topathological evaluation.

Normal breast and breast cancer tissue were collected from patientsundergoing both radical and partial mastectomies. These samples wereplaced in 4 times SDS sample buffer and stored at −20° C. in preparationfor analysis by SDS-PAGE and Western Blotting.

Peripheral blood lymphocytes were obtained during normal serologicaltesting of patients with suspected primary tumours, and during follow-upfor the detection of secondary tumours after treatment.

Collection of Urine

Urine samples were collected from patients diagnosed with breast cancerand also patients found after investigation not to have cancer. Theurine samples were stored at −20° C. until required. The samples wereused in SDS-PAGE without dilution.

Preparation of ASC-Probes

Single cell suspensions were prepared from all available lymph nodes,using standard techniques. Cells were cultured in 2 ml Costar wells or10 ml Falcon flasks under standard culture conditions for up to 10 days.Cell culture supernatants, designated ASC-probes, were harvested aftercentrifugation to remove lymphocytes, and stored at −20° C. until used.

Detection of Cancer-Associated Antigens Using ASC-Probes

Lymphocytes were isolated from lymph nodes draining the breast removedduring surgery being carried out on patients with breast cancer. Tumoursat different stages of growth were also obtained from these patients,and classified according to normal pathological criteria. Normal breasttissue was also obtained.

Initial studies on antigen identification were conducted using aconventional Western blotting technique. Normal and cancerous tissueswere solubilized in SDS-sample buffer and proteins separated onone-dimensional SDS-PAGE. Following electrophoretic transfer tonitrocellulose membranes (Western blotting), the blots were incubatedwith IgG, which had been purified from ASC-probes and biotinylated.Control IgG which was purified from human serum and biotinylated wasalso used to probe similar blots. Bound antibodies were detected afterreaction with peroxidase-conjugated streptavidin and diaminobenzidinedevelopment or enhanced chemiluminescence (ECL). The method isillustrated schematically in FIG. 1.

The relationship between ASC-probe positive bands and knowncancer-associated antigens was examined by stripping the blots of thebound antibodies using standard procedures, and re-probing withmonoclonal antibodies directed against various known cancer-associatedproteins. SDS-PAGE gels were stained for proteins using the Coomassieblue and silver staining techniques.

Additional studies include surface staining of tumour cells, membranepreparations and immunoprecipitation.

EXAMPLE 1

Preparation of ASC-Culture Supernatants

Lymph nodes draining the breast were collected after surgery and wereteased gently in Dulbecco-modified Eagle's medium (DME) containing 10%heat-inactivated fetal calf serum (FCS) and antibiotics (400U/mlpenicillin and 0.1 mg/ml streptomycin). Cells were collected, washedthree times and resuspended to a final concentration of 5×10⁶ cells/mlin complete culture medium (DME supplemented with 400U/ml penicillin,0.1 mg/ml streptomycin, 10% FCS, 2 mM glutamine). Pokeweed mitogen(Sigma Aldrich) was added at a concentration of 2.5 μg/ml to increaseantibody secretion. Culture flasks containing 10 ml cell suspensions or24-well culture plates containing 2 ml cell suspension per well wereincubated at 37° C. in an atmosphere of 5% CO₂ in air, and cellsupernatants harvested 5-6 days later. Culture supernatants containingantibodies secreted by antibody-secreting cells (ASC) present in thelymph node cell suspensions were subsequently stored at −20° C. untilrequired.

EXAMPLE 2

Purification and Labelling of Antibodies from Cell Supernatants

Antibodies secreted by the ASC in culture supernatant were purified byaffinity chromatography using a Protein-G column (Pharmacia HiTrapAffinity Column, 1 ml). The column was pre-equilibrated with loadingbuffer (20 mM sodium phosphate, pH 7.0). The cell supernatants wereapplied to the Protein G column, and the unbound proteins removed using10 column volumes of loading buffer. Bound proteins were eluted from thecolumn using 3 column volumes of elution buffer (0.1 Mglycine/hydrochloric acid, pH 2.7). The eluate was neutralised using 100μl 5 M tris/hydrochloric acid, pH 8.0. The purified antibody solutionwas dialysed extensively against phosphate buffered saline (PBS) beforebeing stored at 4° C.

Control antibodies were purified from the serum of a patient who wasfound not to have breast cancer. The method of purification wasidentical to that used for the ASC supernatants.

Biotinylation of Purified Antibodies

Purified antibodies (Ab) were incubated with a 0.45 mg/ml (finalconcentration) of biotin succinimide ester (Biocap NHS Reagent,Calbiochem, California) in DMSO for 2 hours at room temperature. Thereaction was stopped by overnight dialysis against PBS. Biotinylatedantibodies were then stored at 4° C. until required.

EXAMPLE 3

Western Blotting

Appropriate dilutions of the antigens were loaded on a 10% SDS-PAGE geland blotted on to nitrocellulose membrane (MSI, Melbourne, Australia).Western blot transfers were performed using the wet transfer apparatus(Bio-Rad Laboratories) at a constant voltage of 100V for 1 hour intransfer buffer (25 mM Tris-HCl pH 8.3, 192 mM glycine and 15% (v/v)methanol). After electrophoretic transfer, the membranes were placed ina blocking solution of 3% skim milk powder in PBS for 1 hour, thenwashed 3 times with 0.05% Tween 20 in PBS before being probed withantibody. For urine samples, Western blots were probed for 2 hours withundiluted ASC supernatant, followed by incubation for 1 hour withperoxidase-conjugated rabbit anti-human IgG serum (DAKO) at a 1:2000dilution.

Western blots of breast tissue gave high reactions with the conjugate,due to contamination of the tissue with serum immunoglobulins. Aone-step 2 hour incubation with purified biotinylated antibodies wastherefore used for these tissues, followed by a 1 hour incubation withperoxidase-conjugated streptavidin (Santa Cruz, USA). All incubationswere at room temperature, and all washes were in 0.05% Tween 20 in PBS.Biotinylated antibodies were diluted in 0.5% Tween 20 in PBS containinga dilution of 1 in 50 of non-specific human IgG. Biotinylatedcancer-specific antibodies were used at 1 in 150 dilution, andbiotinylated control antibodies were used at a 1 in 500 dilution. Thesedilutions yielded comparable levels of detection by ECL after westernblotting and reaction with peroxidase-conjugated streptavidin, as shownin FIG. 2.

After reaction with peroxidase conjugates, the membranes were washedagain and then incubated for 1 min in ECL reagent (Amersham PharmaciaBiotech, Sweden) for breast tissue Western blots. Western blots fromurine samples were developed with 3,3′-diaminobenzidinetetrahydrochloride (0.5 mg/ml) (DAB, Sigma Aldrich Pty. Ltd., Australia)in citrate buffer (pH 5.0, 10 mM) to which 0.02% (v/v) hydrogen peroxide(H₂O₂) had been added. When bands were clearly visible the reaction wasstopped with several washes of distilled water.

As shown in FIG. 3, consistently higher background reactivity wasobserved on Western blots of cancer tissues compared to control breasttissue (lane 2 versus lane 4), although both tissues showed similarprotein loading. On Western blots of cancer tissue, several antigens orantigenic regions were clearly reactive with the specific antibodypurified from ASC culture supernatants (lane 1) which were notrecognized by control antibody (lane 2) and were not present in normaltissue (lane 3). The tumour antigens detected in breast tumour tissuehad molecular weights of between approximately 20 and 25 kDa and 40 and60 kDa (arrows).

Urine Antigens

A low molecular weight antigen was recognized by ASC culture supernatantin each of 2 urine samples from breast cancer patients, as shown in FIG.3, lanes 1 and 2. This antigen was not detected in any of the 5 controlurine samples examined (eg lanes 3 and 4).

EXAMPLE 4

Characterisation and Purification of Cancer-Associated Antigens

The cancer-associated antigen identified using ASC-probes arefractionated and purified using conventional analytical and biochemicaltechniques, such as high performance electrophoretic chromatography,capillary electrophoresis, antibody affinity chromatography,immunoprecipitation and high performance liquid chromatography. Oncepurified, the biochemical and biophysical characteristics of theantigens are determined, using standard techniques, including amino acidsequencing. Once sufficient sequence information is obtained, the geneencoding the antigen can be isolated using methods well known in theart, and used to produce recombinant antigen. Alternatively the aminoacid sequence information can be used to make synthetic antigen orfragments thereof, using well-known solid phase synthesis methods. Ifthe antigen is non-protein in nature, for example a carbohydratestructure, other methods of synthesis known in the art may be used. Forexample, solid phase and solution phase synthesis for oligosaccharideshave been described by Alchemia Pty Ltd (PCT/AU01/00054; PCT/AU98/00131;PCT/AU98/00808; PCT/AU01/00028).

The purified, synthetic or recombinant antigen(s) can be used inclassical diagnostic assays, such as ELISA assays, to detect antibodiesin the serum of patients, as shown in FIG. 5.

EXAMPLE 5

Evaluation of Peripheral Blood ASC-Probes for Cancer Diagnosis

Once cancer-associated antigens have been identified using ASC-probesand purified, cloned, or synthesised, they are used to detectcirculating ASCs in peripheral blood of patients at different stages ofdisease, using methods such as ELISA and Western blotting. This isillustrated schematically in FIG. 6. The results are compared to thoseobtained using standard tumour detection methods and serum antibodyresponses. In addition, investigations are initiated to detect the earlyappearance of specific ASCs in peripheral blood of high-risk patientswho are undergoing regular follow-up checks for tumour re-appearanceafter primary therapy.

EXAMPLE 6

Diagnostic Evaluation of Peripheral Blood and Urine for ASC-ProbePositive Antigens

Once cancer-associated antigens have been identified using ASC-probes,the purified, cloned or synthesised antigens are used to develop bothpolyclonal, monoclonal and/or synthetic antibodies. The specificantibodies are used to detect circulating cancer-associated antigen inperipheral blood of patients at different stages of disease, usingmethods such as ELISA and Western blotting. The specific antibodies arealso investigated in a similar manner for use in detectingcancer-associated antigens in the urine of patients at various stages ofdisease, which would enable a non-intrusive diagnostic test. This isillustrated schematically in FIG. 7.

EXAMPLE 7

Soluble and Insoluble Fractions from Breast Tissue

Soluble and insoluble fractions were generated from normal breast tissueand breast tumour tissue by five cycles of freeze-thawing in 20 mMTris-HCl, pH8.0. The samples were centrifuged to pellet the insolublecomponents, followed by analysis of each fraction using 10%SDS-polyacrylamide gel electrophoresis under non-reducing conditions.Following Western blot transfer, the samples were analysed usingbiotinylated IgG purified from ASC probes generated from breast tumourtissue as described in examples 1 and 2. Samples from three individualpatients, designated Patients A-C, were analysed.

As shown in FIG. 8, soluble and insoluble fractions probed withbiotinylated ASC immunoglobulins revealed several regions of activity inboth the normal and tumour tissue. Results from the patient whose tissuewas used to generate the ASC probe are not shown, because normal breasttissue could not be obtained from this patient.

Various differences between the normal and tumour tissue in both thesoluble and insoluble fractions were identified. In the solublefraction, a region at approximately 70 kDa is reactive in both thenormal and the tumour tissue. The major difference between tumour tissueand normal tissue is seen at approximately 64 kDa, with strongreactivity in the tumour tissue but not in the normal tissue. In theinsoluble fraction the differences are not as prominent: however, thetumour samples appear to have a region of activity above 98 kDa whichdoes not appear in the normal tissue.

EXAMPLE 8

Soluble and Insoluble Fractions From Ovarian Tissue

Soluble and insoluble fractions were prepared from normal ovarian tissueand ovarian tumour tissue, and analysed by Western blotting as describedin Example 7. Samples from two individual patients, designated Patient Aand Patient B respectively, were analysed. ASC-probes generated fromPatient A were used.

As shown in FIG. 9, Western blots of the soluble fraction of normaltissue from Patient A detect two main reactive areas, at approximately70 and 120 kDa. In the soluble fraction of tumour tissue of the samepatient, additional strong bands were identified between 70 and 150 kDa,with weaker reactivity being detected between 36 and 50 kDa. In thesoluble fraction from Patient B (tumour, lane 3), the same regions ofreactivity were seen, but the bands were less intense. The insolublefraction from normal tissue showed one area of reactivity atapproximately 70 kDa. The insoluble fraction from tumour tissue ofPatient A showed strong reactivity above 60 kDa, as well as bands ofreactivity at approximately 36 and 50 kDa. The insoluble fraction of thetumour tissue from Patient B showed strong reactivity at approximately70 kDa and 36 kDa.

EXAMPLE 9

Soluble and Insoluble Fractions from Prostate Tissue

Soluble and insoluble fractions were prepared from normal prostate andprostate tumour tissue and analysed by Western blotting as described inExample 8. Samples from four individual patients, designated PatientsA-D, were analysed. ASC-probes generated from Patient A were used.

As shown in FIG. 10, soluble and insoluble fractions probed withbiotinylated ASC immunoglobulins revealed several regions of reactivityin both the normal and the tumour tissues. It should be borne in mindthat tumour tissue will usually include significant amounts of normaltissue, whereas normal tissue would be expected to include very few orno tumour cells. However, there may be some tumour cells in the normalsample, particularly in the case of prostate cancer, because thehyperplastic nature of the whole prostate at the time of collectiongenerally makes it difficult to distinguish the true normal tissue fromthe hyperplastic tissue and borderline tumour tissue. Accordingly, bandswhich are present in the tumour tissue but not in the normal tissue areconsidered to be potential tumour antigens.

Various differences between the normal and tumour tissue were identifiedin both the soluble and insoluble fractions, as indicated by thebrackets. Two main reactive areas were seen in the normal and tumoursamples in both the soluble and insoluble fractions, at approximately 30to 40 kDa and 60 to 100 kDa. In the soluble fraction, all the tumourtissue showed strong bands between 60 and 100 kDa, with weakerreactivity being detected between 30 and 40 kDa. In the insolublefraction, all the tumour tissue examined showed strong bands between 60and 100 kDa with the exception of Patient B. Strong reactivity was alsodetected between 30 and 40 kDa in the insoluble fraction of the tumoursamples.

EXAMPLE 10

Immunoprecipitation of Tumour Specific Antigens

If the body is capable of producing specific antibodies totumour-specific antigens, then these antibodies should be present in thetumour itself, and should form complexes with these antigens. We thoughtthat it might be possible to use these antibodies to collecttumour-specific antigens by immunoprecipitation.

Soluble fractions were extracted from both normal ovarian tissue andovarian tumour tissue by five cycles of repeated freezing and thawing ofthe tissue in 20 mM Tris-HCL, pH8.0. Immune complexes were thenextracted from the soluble fractions using Protein A-Sepharose beads(Pharmacia). The soluble fractions were mixed with Protein A-Sepharose,and allowed to bind for 3 hours at room temperature, with constantmixing. The Protein A-Sepharose beads were washed extensively with 20 mMTris-HCL pH8.0 before elution of the complexes with elution buffer (0.1M Glycine-HCl, pH2.5). The eluate was neutralised using 100 μl 5 mMTris-HCl pH8.0.

Eluates were subjected to Western blotting as described in Example 7.Following Western blot transfer, the examples were analysed usingbiotinylated IgG purified from ASC probes generated from the samepatient.

As shown in FIG. 11, protein staining with Coomassie Blue revealedimmunoglobulin purified from tumour samples using Protein A-Sepharose. Aband of higher molecular weight (indicated by an arrow in sample PS(Tu))was also detected; this possibly indicated some of the immune complexes.On Western blots of normal tissue immunoprecipitate, several reactivebands were seen at molecular weights below 50 kDa. However, in thetumour tissue immunoprecipitate, additional bands were identified atapproximately 250 kDa and 55 kDa, and there was also a region ofactivity between 98 and 64 kDa. These results indicate that tumourantigens are able to be collected using this method.

EXAMPLE 11

F(ab) Blocking of Endogenous Immunoglobulin

The major difficulty in detecting candidate tumour antigens by Westernblot analysis using ASC probes is a consequence of the use ofperoxidase-conjugated antibodies against human immunoglobulins for thedetection of the antigens. All tissue samples, whether from tumours orfrom normal tissue, will include significant amounts of blood, andtherefore will contain human immunoglobulins such as IgGs, the greatmajority of which are directed to antigens other than tumour antigensand are therefore irrelevant to the detection of tumour antigens. Thepresence of this IgG creates a large background of non-specific signalsin Western blots.

In addition to the purification and direct biotinylation of antibodiesas described above, there are at least two alternative strategies whichmay be used to overcome this problem. The first utilises a preliminarystep of blocking endogenous IgG in the sample with F(ab) fragments.

FIG. 12 illustrates schematically how endogenous IgG from blood in thetissue sample may be “blocked” from detection by incubating the blotswith monomeric F(ab) fragments of antihuman IgG (Nielsen et al., 1987;Louis Carl et al., 1993), prior to probing the membrane withperoxide-conjugated anti-human IgG antibodies of the same specificity asthat of the F(ab) fragments. In this way, the background may be reducedto manageable levels, thus increasing the chance of detecting specificsignals.

EXAMPLE 12

F(ab) Amplification/Detection System

A second strategy for overcoming the problem of lack of specificityutilises the suppression of background signal. FIG. 13 illustrates thismethod, in which the background caused by endogenous IgG present inblood in the tissue can be suppressed. The ASC antibodies produced intissue culture are incubated with an excess of biotinylated F(ab) andanti-human IgG, followed by an excess of normal human IgG, so that allbiotinylated F(ab) fragments are bound to IgG (Hierck et al., 1994; Funget al., 1992). These complexes are then used to probe the membrane,following by washing and detection with peroxidase-conjugatedstreptavidin, which binds to biotin. Since all the F(ab) anti-human IgGis already bound to IgG, whether in the form of ASC antibodies or normalIgG, any IgG already present on the blot will not be detected.

It will be apparent to the person skilled in the art that while theinvention has been described in some detail for the purposes of clarityand understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this specification.

References cited herein are listed on the following pages, and areincorporated herein by this reference.

REFERENCES

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1. A method for producing an antibody-secreting cell probe (ASC-probe)against a cancer-specific antigen, comprising the steps of: (a)obtaining a biological sample from an animal suffering from a cancer;(b) isolating a population of cells from the biological sample; (c)culturing the cells in vitro in a suitable culture medium, wherein saidcells are not subjected to in vitro fusion; and (d) harvestingantibodies produced by lymphoid cells present in the cell population toproduce an ASC-probe; in which the cancer is a leukemia.
 2. A methodaccording to claim 1, in which the biological sample is from a tissuearea containing lymphoid cells which is close to the site of the cancer.3. A method according to claim 1, in which the biological sample isselected from the group consisting of blood, lymph or lymph node, andbone marrow.
 4. A method according to claim 3, in which the biologicalsample is spleen or bone marrow.
 5. A method according to claim 1, inwhich the biological sample is a draining lymph node.
 6. A methodaccording to claim 1, in which the biological sample is not blood.
 7. Amethod according to claim 1, in which the cells isolated from thebiological sample include B lymphocytes and/or B memory cells.
 8. Amethod according to claim 1, in which the sample is subjected to one ormore processes for enrichment of lymphocytes or B lymphocytes, orsubjected to one or more processes for selectively depleting cellpopulations which suppress antibody secretion.
 9. A method according toclaim 1, in which the antibodies are separated and purified from theculture medium harvested from the cultured cells.
 10. A method ofisolating an antigen associated with a cancer, comprising the steps of:(a) obtaining a tissue or cell sample from a cancer; (b) culturing thecells in a suitable medium; (c) harvesting antibodies produced bylymphoid cells present in the cell population to produce an ASO-probe;(d) reacting a further sample with the ASC-probe produced in (c) todetect at least one antigen associated with a cancer; and (e) isolatingthe antigen detected, in which the antigen is associated with a specificstage of development of the cancer.
 11. A method according to claim 10,in which proteins present in the tissue or cell sample are separated byelectrophoresis, and the separated proteins are optionally transferredto nitro-cellulose, nylon or other sheets.
 12. A method according toclaim 10, in which in step (d) the product produced by reaction of thefurther sample with the ASC-probe is detected using a detection assayselected from the group consisting of Western blotting,immunoprecipitation assay, a radioimmunoassay, an enzyme-linkedimmunoassay, chemiluminescent assay and immunofluorescent assay.
 13. Amethod for purifying a cancer-specific antigen, comprising the steps of:(a) isolating a population of cells from the biological sample; (b)culturing the cells in vitro in a suitable culture medium; (c)harvesting antibodies produced by lymphoid cells present in the cellpopulation to produce an ASC-probe; (d) subjecting a crude antigenmixture present in an extract of cancer cells or cancer tissue toaffinity chromatography using the ASC-probe prepared in step (c),immobilized on a suitable support; and (e) isolating antigen bound tothe immobilized ASC-probe; in which the antigen is associated with aspecific stage of development of the cancer.